Nanoscale imaging of molecular positions and anisotropies

Abstract

A Polarization Fluorescence Photoactivation Localization Microscopy (P-FPALM) system and method are provided to simultaneously image the localizations and fluorescence anisotropics of large numbers of single molecules within a sample. The system modifies known FPALM systems by adding a polarizing beam splitter. The beam splitter polarizes emissions perpendicular and parallel to an axis in the sample to allow spatially separate imaging of fluorescence emitted from a sample. The system includes lenses and mirrors so that the separate, polarized beams are detected simultaneously. The present invention includes methods of using the system to image localizations and fluorescence anisotropics of single molecules, and methods of using data obtained with the system to predict 3-D orientation of the molecules. The system and method achieve substantially improved lateral resolution within even dense samples over known microscopic imaging techniques, and does not compromise speed or sensitivity.

Description

FIELD OF THE INVENTION[0001]The present invention relates to a system and method of imaging at a nanoscale level. More particularly, the present invention is a system and method for imaging single molecule polarization anisotropy in biological specimens.BACKGROUND OF THE INVENTION[0002]For a complete understanding of cellular biology we ideally need to observe the cell at a molecular level. Single-molecule detection gives extra statistical information and allows the exploration of heterogeneity in samples, as well as direct observation of dynamic state changes arising from photophysics and photochemistry. For every distinct molecule it can be useful to know the number of its kind present, the precise location of each member of the ensemble of identical molecules, and the functionality of each member of the ensemble. Such observations can only be made by single molecule microscopy, since standard imaging methods, which make ensemble measurements that average over many molecules, do n...

AI Technical Summary

Benefits of technology

[0008]The system of the present invention incorporates a polarizing beam splitter into the detection path of a standard FPALM microscope. This modification allows simultaneous, spatially separate imaging of the fluorescence emitted by a molecule, and this emission is polarized parallel and perpendicular to the excitation polarization. The present invention also modifies the standard FPALM system by adding lenses which expand the emitted fluorescent image paths after polarization, and additional mirrors which adjust the two detection paths to have the same or nearly the same total length from the beam splitter to the image detector. The method of the present invention analyzes the relative intensities of molecules in the two images to yield the anisotropy of each localized molecule. Two-dimensional maps (images) of single-molecule anisotropy can be obtained with significantly improved spatial resolution.

[0009]In one example of the system and method of the present invention, the sample is placed on the stage of any suitable microscope together with a suitable imaging lens. The use of a water-immersion lens is advantageous because it minimizes aberrations when imaging a sample that is also in water. The sample is illuminated using a light source of suitable wavelength. In one embodiment of the present invention the light source used is a laser. In another embodiment the light source of the microscope system includes two lasers: an activation laser and a readout laser of suitable wavelength. The light source is focused in the objective back-aperture to cause a large area of the sample to be illuminated.

[0010]In one embodiment, illumination using a relatively unfocused Gaussian beam is advantageous because it reduces the tipping of the polarization toward the z-axis which results from a high-numerical aperture diffraction-limited focus. In another embodiment, the intensities of the illumination light source are modulated at one or more wavelengths. This modulation can be accomplished using a mechanical or optical shutter or an electrooptic modulator such as a Pockel's cell. This modulation allows sequences of optical pulses to prepare sample molecules in different photophysical states. In yet another embodiment, polarization of the illumination light source is modulated using mechanical or optical shutters which allow illumination light of different polarizations to pass. In embodiments using two lasers as the illumination light source, either the activation or readout beam or both may be modulated in this way. One embodiment includes splitting the illumination light into two or more separate paths with different polarizations that are independently shuttered. Another embodiment modulates the illumination polarization using an electrooptic modulator such as a Pockel's cell. This modulation will allow molecules with different orientations to be selectively excited.

[0015]The ability to image anisotropy with resolution below the diffraction limit presents several interesting opportunities, most importantly the ability to image short-range order and to quantify the degree of preferential orientation of molecules. As long as the limitations of the method are taken into account, we can use the anisotropy to estimate the degree of alignment (but not the precise angle) between the transition-dipole moment of the emitting molecule (the fluorophore) and the excitation laser beam polarization. Interactions between membrane domains and the cytoskeleton, such as those found in focal adhesions, are expected to result in preferential orientation of molecules, but the size of those structures is generally well below the diffraction limit. The improved resolution in P-FPALM will allow quantification in the order of proteins and lipids in membrane domains at length scales inaccessible to standard methods.

[0016]In addition to its dramatically improved spatial resolution, P-FPALM provides absolute numbers of molecules and can quantify heterogeneous populations of molecules, both which are inaccessible to conventional methods. P-FPALM provides a means to measure molecular positions and orientations in biological structures in a crucial, but previously inaccessible, range of length scales. Furthermore, P-FPALM will be compatible with live-cell FPALM, PALM (photo-activated localization microscopy) and STORM (stochastic optical reconstruction microscopy) using widefield excitation, and with multi-color imaging.

[0019]Longer acquisitions may allow higher molecular densities to be observed in well-immobilized samples. Extension of the technique to three-dimensional (3D) imaging is both possible and useful, considering that structures such as actin will span many focal planes. Hence, 3D imaging would capture larger numbers of total molecules in different focal planes and allow extended structures to be visualized even more comprehensively.

Problems solved by technology

Such observations can only be made by single molecule microscopy, since standard imaging methods, which make ensemble measurements that average over many molecules, do not provide quantitative information about each molecule.

Light microscopy provides non-invasive imaging of multiple species in biological specimens with single-molecule sensitivity, but diffraction limits the resolution to ˜150-250 nm.

However, despite its impressive capabilities, this method does not provide information about the anisotropy and rotational freedom of individual molecules.

Images